title: Stable knockdown of PASG enhances DNA demethylation but does not accelerate cellular senescence in TIG-7 human fibroblasts.
abstract: Demethylation of 5-methylcytosine in genomic DNA is believed to be one of the mechanisms underlying replicative life-span of mammalian cells. Both proliferation associated SNF2-like gene (PASG, also termed Lsh) and DNA methyltransferase 3B (Dnmt3b) knockout mice result in embryonic genomic hypomethylation and a replicative senescent phenotype. However, it is unclear whether gradual demethylation of DNA during somatic cell division is directly involved in senescence. In this study, we retrovirally transduced TIG-7 human fibroblasts with a shRNA against PASG and compared the rate of change in DNA methylation as well as the replicative life-span to control cells under low (3%) and ambient (20%) oxygen. Expression of PASG protein was decreased by approximately 80% compared to control cells following transduction of PASG shRNA gene. The rate of cell growth was the same in both control and PASG-suppressed cells. The rate of demethylation of DNA was significantly increased in PASG-suppressed cells as compared control cells. However, decreased PASG expression did not shorten the replicative life-span of TIG-7 cells. Culture under low oxygen extended the life-span of TIG-7 cells but did not alter the rate of DNA demethylation. While knockout of PASG during development results in genomic hypomethylation and premature senescence, our results show that while downregulation of PASG expression in a somatic cell also leads to DNA hypomethylation, there is no associated senescent phenotype. These results suggest differences in cellular consequences of hypomethylation mediated by PASG during development compared to that in somatic cells.
authors.affiliation: Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, Chuo-ku, Chiba, Japan. piesuke@faculty.chiba-u.jp
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Suzuki
authors.forename: Jason E
authors.initials: JE
authors.lastname: Farrar
authors.forename: Srinivasan
authors.initials: S
authors.lastname: Yegnasubramanian
authors.forename: Muhammed
authors.initials: M
authors.lastname: Zahed
authors.forename: Nobuo
authors.initials: N
authors.lastname: Suzuki
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Arceci
mesh_terms: D000693:Anaerobiosis; D000818:Animals; D001483:Base Sequence; D049109:Cell Proliferation; D016922:Cellular Senescence / Q000187:drug effects / Q000235:genetics*; D004265:DNA Helicases / Q000037:antagonists & inhibitors / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D055785:Gene Knockdown Techniques; D006801:Humans; D051379:Mice; D010100:Oxygen / Q000378:metabolism / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D012190:Retroviridae; D014161:Transduction, Genetic
chemical_list: D034741:RNA, Small Interfering; D004265:DNA Helicases; C412063:HELLS protein, human; D010100:Oxygen